CN105200002A - Sisomicin-producing micromonospora purpurea engineering bacterium, and construction and application thereof - Google Patents
Sisomicin-producing micromonospora purpurea engineering bacterium, and construction and application thereof Download PDFInfo
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Abstract
The invention provides a sisomicin-producing micromonospora purpurea engineering bacterium, and construction and application thereof, and belongs to the field of microbe pharmacy, and clarifies genB4 gene function by means of gentamycin-producing micromonospora purpurea G1008. The gentamycin-producing micromonospora purpurea G1008 is selectively bred, and an original strain Gb taking gentamycin C1a metabolism approach as the main approach is obtained. By constructing shuttle plasmids pFD306 and pGB403 knocking out genK and genB4 and successively introducing the shuttle plasmids pFD306 and pGB403 into micromonospora purpurea Gb and screening zygotes, the sisomicin-producing micromonospora purpurea GB4408 is obtained, and is characterized by possessing the unique heredity characteristic of highly producing sisomicin biosynthesis potential. Construction of the engineering bacterium is not reported at home and abroad, and application to prepare sisomicin antibiotic medicaments is satisfied.
Description
Technical field
The invention belongs to field of microbial pharmacy, relate to and produce the little monospore engineering bacteria of sisomicin magneta colour and structure thereof and application, the present invention utilizes molecular biology manipulations technology, illustrates
genB4the function of gene, and using deep red micromonospora G1008 as starting strain, the sub-strain that to screen with Gentamicin C1a be main metabolic regulation, knocks out
genB4with
genKgene, thus the little monospore engineering bacteria of magneta colour obtaining that a plant height produces sisomicin (Fig. 1), can be applied to sisomicin manufacture.
Background technology
Amino-oligosacchride is important anti-infectives, has important clinical value, unique anti-microbial activity.Through long-term clinical application and the test of over half a century, obtain good prestige, lasting, one of primary categories becoming anti-infectives.Most of amino-oligosacchride medicine contains two to four glycosyls, and each glycosyl is amido modified by one to two, and the amino of whole amino-oligosacchride molecule must between 4-6.Very little, anti-infection activity is on the low side for amino; Amino too many, toxicity is too large, can not meet the requirement of clinical safety.As kantlex, tobramycin, gentamicin, sisomicin, G418, G52, Streptomycin sulphate, ribostamycin, Liu Suanyan NEOMYCIN SULPHATE and lividomycin etc., all meet this primitive rule.
Sisomicin, the amino-oligosacchride such as gentamicin, G418 and G52 class microbiotic, produced by micromonospora.Sisomicin producing strains is Micromonospora inyoensis; Gentamicin, G418 and G52 etc. are mainly purple-red single-spore bacteria and produce.The microbiotic that purple-red single-spore bacteria produces, biosynthesizing potentiality are higher than Micromonospora inyoensis a lot, and required starting material are also relatively universal, and do not have Micromonospora inyoensis so harsh, therefore relative cost is lower, are also convenient to produce Control and management.But Micromonospora inyoensis main product sisomicin.Therefore, industrialization is produced sisomicin and is still adopted Micromonospora inyoensis.If purple-red single-spore bacteria can be obtained produce sisomicin bacterial strain, be then expected to the sisomicin obtaining more high yield, significantly reduce production cost further, produce larger economic benefit and social benefit.
Deep red micromonospora is gentamicin producing strains, and main ingredient is gentamicinC race mixture: C1a, C1, C2 and C2a etc., also has the small component such as G418, G52 and sisomicin in addition.This is for provide biological function basis from purple-red single-spore bacteria artificial constructed product sisomicin engineering bacteria.Along with the development of Protocols in Molecular Biology, provide possibility for building sisomicin producing strains from purple-red single-spore bacteria.Along with the fast development of information biology and genetic engineering technique, for genetically engineered directive breeding is laid a good foundation.Within 2012, we disclose domestic first case gentamicin biological synthesis gene cluster, and at gene pool registration (GenBankaccessionNo.:JQ975418,44181bp).Establishing the deep red micromonospora conjugal transfer system of system simultaneously, for obtaining important gentamicin biosynthetic metabolism intermediate, having established solid foundation.
Although sisomicin has been applied to clinical, industrialization is produced also has had decades, and production cost still remains high.If can obtain more high yield, processing requirement is more flexible, the bacterial strain that adaptability is stronger, and the economic benefit that obvious industrialization produces and social benefit will be better.Therefore, build product sisomicin engineering bacteria from purple-red single-spore bacteria and just seem necessary, very worth research and development.
But difficulty mainly, current, fail so far both at home and abroad all to illustrate all gene functions in gentamicin, sisomicin biological synthesis gene cluster, let alone illustrate their biosynthetic controlling mechanism.Therefore, build from purple-red single-spore bacteria and produce sisomicin engineering bacteria, also just become an international difficult problem.
Summary of the invention
The object of the present invention is to provide and produce the little monospore engineering bacteria of sisomicin magneta colour and structure thereof and application, the present invention is by research purple-red single-spore bacteria series
genBthe function of dependency structure gene, illustrates the key gene relevant with sisomicin biosynthesizing
genB4, in conjunction with traditional breeding way, obtain the starting strain of pathways metabolism based on Gentamicin C1a, on the basis of theoretical investigation, successfully construct the little monospore engineering bacteria of product sisomicin magneta colour of applicable industrialization.This achievement in research has no report both at home and abroad, achieves practicality effect, may be used for sisomicin industrialization and produces.
For realizing above-mentioned target, the present invention adopts following technical scheme:
Content comprises: by illustrating
genB4gene function.And seed selection is carried out to the purple-red single-spore bacteria producing gentamicin, obtain based on the starting strain of Gentamicin C1a pathways metabolism, then deactivation
genB4with
genKgene, obtains the little monospore engineering bacteria of product sisomicin magneta colour that hereditary property is more superior than Micromonospora inyoensis.This Strain Designation is deep red micromonospora GB4408(
micromonosporapurpureagB4408), finish China Committee for Culture Collection of Microorganisms's common micro-organisms center registration guarantor on October 12nd, 2015, No. 3 in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, deposit number is CGMCCNo.11483.
The present invention illustrates
genB4gene function is block the conversion from Sisomicin to Gentamicin C1a and the conversion from VDM to gentamicinC2, and its mechanism is the two dehydrogenations participating in gentamicin deep red brown sugar amine C4 ' and C5 '.With deep red micromonospora G1008 for starting strain, through physics and chemistry factor treatment, conjugated metabolite structure is determined, obtains main product Gentamicin C1a sub-starting strain purple-red single-spore bacteria Gb, for gene recombination, build and produce the little monospore engineering bacteria of sisomicin magneta colour.
The present invention, by screening, obtains the sub-strain of high yield Gentamicin C1a, and using these subspecies as starting strain, through gene knockout, in deactivation deep red micromonospora
genB4with
genKgene, obtains the little monospore engineering bacteria of magneta colour producing sisomicin, mainly comprises following step:
A. illustrate
genB4gene function;
B. Gentamicin C1a superior strain is screened;
C. build and knock out
genB4with
genKgene shuttle vectors;
D. shuttle vectors imports deep red micromonospora;
E. zygosporic screening;
F. the qualification of sisomicin engineering bacteria is produced.
The invention has the advantages that: produce sisomicin with purple-red single-spore bacteria, required raw material sources is convenient, inexpensive, can greatly reduce fermentative production cost; Block the biosynthesizing of gentamicinC1 pathways metabolism, thoroughly cut off the synthesis of each component of this pathways metabolism structural similitude, facilitated the Hydrolysis kinetics of sisomicin, greatly reduced extraction production process; Without the need to chromatographic separation, just can obtain highly purified sisomicin, reach the modern production requirement of environmental type; Obtain the bacterial strain of main product Gentamicin C1a through screening, as the starting strain that genetically engineered is optimized, after genetic modification, sisomicin productivity is still very high, meets industrialized requirement; In the world, the function of genB4 gene is illustrated first.
Accompanying drawing explanation
Fig. 1: sisomicin (sisomicin) chemical structural formula.
Fig. 2 plasmid pGB403 restriction enzyme digestion and electrophoresis detects figure, 1:DL5000Marker; 2:
xbai/
ecor I; Wherein A figure is pGB403 plasmid; B figure is pGB403 plasmid enzyme restriction electrophoresis detection figure.
The zygosporic PCR Testing and appraisal of Fig. 3 GB4408: 1:DL5000Marker; 2:G1008 (P5/P6); 3:GB41 (P5/P6); 4:GB4408 (P5/P6); Wherein A figure is deactivation genB4 gene schematic diagram; B figure is the PCR detection figure knocking out genB4 gene.
The HPLC detected result of Fig. 4 GB4408 meta-bolites: wherein A figure is that starting strain G1008 meta-bolites compares; B figure is the HPLC detected result of GB4408 meta-bolites.
Fig. 5 GB4408 engineering bacteria meta-bolites mass spectrometry results; Wherein A figure is that starting strain G1008 meta-bolites compares; B figure is the mass spectrometric detection result of GB4408 meta-bolites.
Fig. 6 GbKB4 fermentating metabolism product mass spectroscopy figure; Wherein A figure is the mass spectrometric detection result of GB4408 meta-bolites, compares; B figure is the mass spectrometric detection result of GbKB4 engineering bacteria meta-bolites.
Fig. 7 GbKB4 engineering bacterium fermentation meta-bolites HPLC analytical results.
Embodiment
Embodiment 1
Gene
genB4illustrating of function:
With the gentamicin biological synthesis gene cluster of registering at Genebank (accession number: JQ975418) for template, bioinformatics software VectorNTI11.5 is utilized to design two pairs of primers, amplification
genB4gene upstream and downstream sequence is as Homo~logous exchange arm: primer P1/P2 for increase upstream exchange arm B41, primer P3/P4 for the downstream exchange arm B42 that increases.Design primer P5/P6 is used for
genB4the detection of gene disruption mutant strain.As shown in Figure 2, primer sequence and restriction enzyme site thereof are in table 1 for primer and expanding fragment length.
Table 1 gene
genB4functional study relevant primer
the structure of 1 recombinant plasmid pGB403
Extract purple-red single-spore bacteria G1008 genomic dna, as template.Pcr amplification
genB4upstream and downstream exchange arm B41 and B42.Two exchange arms are cloned into pMD19-T carrier respectively, obtain middle interstitial granules pGB401 and pGB402.Plasmid pGB401 uses
hind III He
ecor I enzyme is cut, and reclaims 1963bp fragment; Plasmid pGB402 uses
hind III He
xbai enzyme is cut, and reclaims 2019bp fragment; PKC1139 uses
xbai He
ecor I enzyme is cut, and reclaims 6446bp fragment.Connected by above three fragment enzymes, enzyme connects sample and transforms Top10 competent cell, screening positive clone, final acquisition recombinant plasmid pGB403.With
ecor I He
xbai couple of pGB403 carries out enzyme and cuts, and can obtain 6446bp and 3994bp two band in theory.Enzyme cuts sample through electrophoresis, two bands detected, has conformed to theoretical prediction, finally by order-checking, proves that plasmid pGB403 is target plasmid (Fig. 2).
2GB4408
the structure of bacterial strain
Recombinant plasmid pGB403 is transformed
e.colieT12567(pUZ8002) conjugal transfer donor bacterium is obtained
e.colieT12567(pGB403/pUZ8002).Using the spore of purple-red single-spore bacteria as conjugal transfer recipient bacterium.According to conjugal transfer method, mixed by spore suspension with donor bacterium, dilution spread, after plate culture medium, cultivates about 10h for 37 DEG C, covers, continue cultivation one week, until grow zygote on flat board with containing apramycin and naphthyridines aqueous acid.
Choose the good zygote of a strain growing way, extract genomic dna, carry out pcr amplification detection with P5/P6 primer.Obtain two bar segment, be respectively 1241bp and 512bp, prove that it is that engineering bacteria is changed in single cross.PCR primer electrophoresis detection result conforms to expection (Fig. 3 B), show recombinant plasmid pGB403 Successful integration to purple-red single-spore bacteria G1008, called after micromonospora GB41.Fig. 3 A is shown in by plasmid pGB403 and G1008 homologous recombination schematic diagram.
Single cross is changed bacterial strain GB41 in 37 DEG C, containing cultivation 3 generation lax on antibiotic inclined-plane, be then separated single bacterium colony, screened by single bacterial colony photographic reprinting point plate method.From 408 strain bacterial strains, obtain 2 strains not long in resistant panel, at the single bacterium colony without normal growth on medicine flat board, choose the bacterial strain that a wherein strain is grown fine, extract genomic dna, utilize double exchange primers designed P5/P6 to carry out PCR detection.Result detects the fragment of 512bp and 1241bp, thinks double exchange engineering bacteria, as Fig. 3 B.Be purple-red single-spore bacteria GB4408 by this Strain Designation, be called for short GB4408.PCR primer checked order, result proves that GB4408 engineering bacteria is purpose project bacterium really.
metabolite is analyzed
The fermentation liquor 732-NH of GB4408
3 +resin absorption, 5% ammoniacal liquor is resolved, and after the process such as alcohol settling, obtains Raw samples, carries out TLC analysis, and compared with standard substance, result is the not gentamicinC Group Component such as resynthesis gentamicinC1, C1a, C2, then other gentamicin intermediate of synthesis.
Carry out HPLC analysis to GB4408 meta-bolites, detected result is shown in Fig. 4, and the retention time of each component of gentamicin standard substance is respectively: C1(5.98min), C1a(14.58min), C2a(18.57min), C2(21.05min); There are 3 main peaks in the Raw samples of GB4408, retention time is respectively 7.68min, 18.72min and 20.12min, and none conforms to the retention time of gentamicin standard substance.The meta-bolites of further proof GB4408 does not have gentamicinC Group Component.
The component of GB4408 meta-bolites, analyzes through MS, the results are shown in Figure 5.Leading ion peak has 4, is respectively 448.3(Sisomicin), 462.3(VDM), 476.3(VDM C6 ' position methylate), 497.3(G418) four kinds of components.231.6 and the 249.1 double charge peaks being respectively VDM and G418,322.2 is fragment peak.
4 genes
genB4illustrating of function
These results suggest that,
genB4the inactivation of gene, has blocked the conversion from Sisomicin to Gentamicin C1a and the conversion from VDM to gentamicinC2, infers
genB4gene participates in two dehydrogenations of gentamicin deep red brown sugar amine C4 ' and C5 '.
embodiment 2
the seed selection of Gentamicin C1a superior strain
With gentamicin producing strains G1008 for starting strain, through ultraviolet mutagenesis, single bacterium colony separation and purification, fermentation, extracts, and meta-bolites structure is determined to wait detection, determines the mutant strain that Gentamicin C1a component progressively improves.Through 48 generations, directive breeding more than 3 years, obtain main product Gentamicin C1a sub-starting strain purple-red single-spore bacteria Gb, for next step gene recombination, structure product single-component sisomicin engineering bacteria.
embodiment 3
GbKB4
the structure of engineering bacteria
According to the method for similar embodiment 1, by this research of plasmid pFD306(series of patents: 201110331534.9) import donor bacterium, obtain donor bacterium
e.colieT12567/(pFD306/pUZ8002).The spore of donor bacterium and purple-red single-spore bacteria Gb is carried out conjugal transfer, after 7 days, MS flat board grows zygote, the strain that picking grows fine, called after GbK.Be forwarded to micromonospora seed culture medium, start to be separated single bacterium colony after lax cultivation 4 generation, and photocopy is to containing 50 μ/mL apramycins with not containing on antibiotic flat board, screens and obtains engineering bacteria GbK(△
genK), extract its genomic dna, carry out PCR checking with primer P5/P6, electrophoresis detection and DNA sequencing, confirm as purpose project bacterium.
△ is knocked out according to similar above
genKmethod, plasmid pGB403 is imported donor bacterium
e.colieT12567, obtains donor bacterium
e.colieT12567/(pGB403/pUZ8002).By donor bacterium ET12567/(pGB403/pUZ8002) and GbK(△
genK) spore carry out conjugal transfer, after 7 days, MS flat board grows zygote, the strain that picking grows fine, called after GbKB4-1.Be forwarded to micromonospora seed culture medium, start to be separated single bacterium colony after lax cultivation 4 generation, and photocopy is to containing 50 μ/mL apramycins and not containing on antibiotic flat board, screening obtains magneta colour little monospore engineering bacteria GbKB4(and is called for short GbKB4), extract its genomic dna, carry out PCR detection with primer P5/P6, DNA sequencing, result and theory speculates coincide, and are purpose project bacterium.
Embodiment 4:
engineering bacterium fermentation and product extraction and analysis
The fermentation of 1 engineering bacteria GbKB4
Seed culture medium: glucose 0.5%, W-Gum 1.0%, Semen Maydis powder 1.5%, peptone 0.2%, soybean cake powder 1.0%, KNO
30.05%, CaCO
30.5%, pH7.0.
Fermention medium: starch 6.0%, Semen Maydis powder 1.0%, peptone 0.4%, groundnut meal 2.0%, NaNO
30.01%, NH
4cl0.1%, CaCO
30.5%, pH7.5.
The fermentation of purple-red single-spore bacteria GbKB4.Engineering bacteria GbKB4 transfers slant medium, cultivates 10 days at 37 DEG C, treats that slant pore is ripe, with inoculating needle scraping 1cm2 spore to seed culture medium.Under 280rpm/min condition, on 37 DEG C of shaking tables, cultivate 40 hours, when deep cultivation enters logarithmic phase, transfer in fermention medium (loading amount is 40mL/250mL triangular flask) by 5% inoculum size, 35 DEG C of shaker fermentation 120h (rotating speed is 280rpm).
20000 liters of fermentor tanks are produced, mixing speed 180 revs/min, air flow 1:0.5 ~ 0.8 (M
3/ M
3min), substratum, culture temperature, inoculum size ratio, fermentation time etc., are similar to shake flask fermentation, put tank fermentation unit and reach 1728 μ/ml.
2 meta-bolitess extract
A, slightly to carry
Acidifying after fermented liquid dilution, after alkali adjusts back pH6.8, uses 732-NH
4 +resin Static Adsorption 4 hours.Collect absorption saturated resin, with 0.01MHCl solution pickling saturated resin, then with deionized water washing to neutral, then carry out alkali cleaning with pH7.5 ammoniacal liquor, alkali cleaning volume is no less than 12 times of saturated resin volume.Then be connected in series on isopyknic 711 resin columns, collect the de-liquid of tree.
Proximate analysis, adopts mass spectrometric detection, the results are shown in Figure 6.Different from GB4408 meta-bolites, GbKB4 engineering bacteria leading ion peak is 448 [M+H]
+, 470 [M+Na]
+, 486 [M+K]
+with Sisomicin molecular weight homogeneity.Secondary quasi-molecular ions is 483 [M+H]
+with mesostate gentamicinX 2 homogeneity.242 [M+H]
+for gentamicinX 2 double charge peak, other peak is fragment peak.
B, refining
Elutriant to about 300000ug/mL, is adjusted to pH5.5 ~ 6.0 with the vitriol oil through thin film concentration, and add 5% activated carbon decolorizing, transparence reaches more than 92%, filters and removes solids, obtain transparent settled solution.Under agitation, slowly in concentrated solution drip more than 95% ethanol, carry out crystallise overnight, after through centrifugation, namely 85% ethanolic soln drip washing obtain wet finished product.Through vacuum-drying (more than vacuum tightness 500mmHg, temperature 60
0c, dry 6 hours), obtain meta-bolites highly finished product.
engineering bacteria GbKB4 Methanogenesis
Engineering bacteria GbKB4 fermentating metabolism product, analyzes through HPLC, the results are shown in Figure 7.The moving phase used with reference to detection gentamicin is analyzed, and the retention time that Sisomicin goes out peak is 18.64min, confirms as Sisomicin.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCELISTING
Rong De bio tech ltd, Gulou District, <110> Fuzhou City
<120> produces the little monospore engineering bacteria of sisomicin magneta colour and structure thereof and application
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Claims (4)
1. produce the little monospore engineering bacteria of sisomicin magneta colour, it is characterized in that: described engineering bacteria is for being deep red micromonospora GB4408(
micromonosporapurpureagB4408), finish China Committee for Culture Collection of Microorganisms's common micro-organisms center registration guarantor on October 12nd, 2015, deposit number is CGMCCNo.11483.
2. the structure of the little monospore engineering bacteria of main product sisomicin magneta colour as claimed in claim 1, it is characterized in that: with deep red micromonospora G1008 for starting strain, through physics and chemistry factor treatment, conjugated metabolite structure is determined, obtain main product Gentamicin C1a sub-starting strain purple-red single-spore bacteria Gb, for gene recombination, build and produce the little monospore engineering bacteria of sisomicin magneta colour.
3. the structure of product sisomicin engineering bacteria according to claim 2, is characterized in that, by gene substitution, removes in deep red micromonospora Gb
genKwith
genB4gene, blocks gentamicin biosynthesizing, ends at sisomicin site, mainly comprise the following steps:
A. illustrate
genB4gene function;
B. Gentamicin C1a superior strain is screened;
C. build and knock out
genB4with
genKgene shuttle vectors;
D. shuttle vectors imports deep red micromonospora;
E. zygosporic screening;
F. the qualification of sisomicin engineering bacteria is produced.
4. the little monospore engineering bacteria of sisomicin magneta colour that produces as claimed in claim 1 is preparing the application in sisomicin antibiotic medicine.
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| CN110358718A (en) * | 2019-07-19 | 2019-10-22 | 福州市鼓楼区荣德生物科技有限公司 | The building and its application of main product Gentamicin C1a engineering bacteria |
| CN112553354A (en) * | 2020-12-24 | 2021-03-26 | 福安药业集团烟台只楚药业有限公司 | Molecular biological method for rapidly detecting gentamicin high-yield strain |
| CN112725257A (en) * | 2021-03-03 | 2021-04-30 | 莆田学院 | GMRA1,2,3 compound and biosynthesis and application thereof |
| CN114369126A (en) * | 2022-01-19 | 2022-04-19 | 福州市鼓楼区荣德生物科技有限公司 | Biosynthesis and application of GMRA11,12 and 13 compound |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358718A (en) * | 2019-07-19 | 2019-10-22 | 福州市鼓楼区荣德生物科技有限公司 | The building and its application of main product Gentamicin C1a engineering bacteria |
| CN112553354A (en) * | 2020-12-24 | 2021-03-26 | 福安药业集团烟台只楚药业有限公司 | Molecular biological method for rapidly detecting gentamicin high-yield strain |
| CN112725257A (en) * | 2021-03-03 | 2021-04-30 | 莆田学院 | GMRA1,2,3 compound and biosynthesis and application thereof |
| CN114369126A (en) * | 2022-01-19 | 2022-04-19 | 福州市鼓楼区荣德生物科技有限公司 | Biosynthesis and application of GMRA11,12 and 13 compound |
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